Enterohemorrhagic Escherichia coll (EHEC) are an emerging group of food- and water-borne pathogens that colonize the distal ileum and colon and produce potent cytoxins. EHEC infection causes illnesses ranging from mild diarrhea to the potentially fatal hemolytic uremic syndrome. These bacteria, particularly serotype O157:H7, represent a significant threat to public health and are classified as NIAID Category B Priority pathogens. The O157:H7 genome contains -1.4 megabases of DMAthat are not present in the genome of non-pathogenic E. coll K-12. This additional DMA is interspersed as 177 'O-islands' along the backbone of sequences that are shared with E. coll K-12. Our central hypothesis is that the O-islands encode the majority of the pathogenic properties of E. coli O157:H7. All the reported EHEC virulence factors are encoded in O-islands, and we recently identified an additional O-island encoded factor, AcfO, that promotes EHEC intestinal colonization. We have developed methodology to rapidly and comprehensively survey the contribution of uncharacterized O-islands to EHEC pathogenicity. Although the interaction of E. coli O157:H7 with mammalian cells in vitro has been extensively studied, relatively little is known about the EHEC factors that are required for intestinal colonization. This gap in our knowledge is primarily due to the lack of a readily available animal model of human EHEC infection. Our preliminary data demonstrates that infant rabbits are an excellent model host for study of the intestinal manifestations of E. coli O157:H7 infection. Infant rabbits develop diarrhea and colitis, and quantitative analysis of intestinal colonization can be performed. In this proposal, we will utilize infant rabbits to carry out a comprehensive, high throughput screen of EHEC mutants with single O-islands deleted. In Aim I, we will identify the EHEC O-island genes that promote intestinal colonization. Our approach for investigating the mechanisms of action of gene products identified here is illustrated by the experiments proposed in Aim II, where we will characterize the expression, regulation, function, transmission and distribution of acfO. These studies will yield valuable new information about the mechanisms of EHEC pathogenicity and identify colonization factors that may provide new targets for novel therapeutics and vaccines.